Method and Apparatus for Decellularization of Tissue

ABSTRACT

Methods of decellularization of tissue, such as mammalian tissue, are provided, along with methods of making an extracellular matrix (ECM) preparation. Systems and apparatus useful in performing the methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/123,471 filed Sep. 2, 2016, which is a national stage ofInternational Patent Application No. PCT/US2015/018744, filed Mar. 4,2015, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/947,578, filed Mar. 4, 2014, each of which is incorporated hereinby reference in its entirety.

BACKGROUND

Methods of decellularization of tissue, such as mammalian tissue, areprovided, along with methods of making an extracellular matrix (ECM)preparation. Systems and apparatus useful in performing the methods arealso provided.

ECM materials have found broad use in the field of regenerativemedicine. A variety of methodologies for the preparation of ECMmaterials exist, occasionally meeting with success, as evidenced by themany commercial products available. However, there are limitations tothe methods, such as ECM materials with inferior mechanics, such asfailure stress and stiffness as compared to the mechanical qualitiesactually needed in many instances. Further, traditional methods do notnecessarily work on certain tissue types and can be overly destructiveto the ECM structure, resulting in ECM material that is useless orsub-optimal for a desired end use.

Tracheal defects or stenosis can result from congenital defects, trauma,or various pathologies such as cancer or infection. Partial loss ofairway in a patient is debilitating and life-threatening. In pediatricpatients surgical approaches including, slide tracheoplasty has beenemployed successfully. In adults trachea mobilization has enabledpost-resection repair in some cases. Long-term stenting, dilation, andtracheostomy have been employed as palliative care in some patients.However, regardless of the approach complication rates remains very highand long term morbidity is common. There remains a cohort of patientsfor which standard approaches cannot be employed. Therefore, afunctional tracheal replacement graft is still desirable.

Initially, engineered tracheal grafts were consisted of purifiedcollagen sponges around a stent or synthetic scaffold (Okumura, N., etal., Experimental study on a new tracheal prosthesis made fromcollagen-conjugated mesh. J Thorac Cardiovasc Surg, 1994. 108(2): p.337-45 and Teramachi, M., et al., Intrathoracic tracheal reconstructionwith a collagen-conjugated prosthesis: evaluation of the efficacy ofomental wrapping. J Thorac Cardiovasc Surg, 1997. 113(4): p. 701-11).Though widely used, these have had multiple deficiencies. Failure of thefirst engineered tracheas resulted from several causes includinginfection, stenosis, and complete disintegration (Wurtz, A. and E.Kipnis, Tissue-engineered airway in the clinical setting: a call forinformation disclosure. Clin Pharmacol Ther, 2012. 91(6): p. 973; authorreply 974). Current engineered tracheas are considerably more complexand employ both multiple graft modifications and recipient treatments.Common to all is a foundation built upon a decellularized trachealallograft or a synthetic nanofiber scaffold to provide structuralsupport. These scaffolds are then seeded with mature airway cells orstem cells and transplanted to recipients pre-treated with growthfactors (Gilbert, T. W., et al., Decellularization of tissues andorgans. Biomaterials, 2006. 27(19): p. 3675-83 and Gilbert, T. W.,Strategies for tissue and organ decellularization. J Cell Biochem, 2012.113(7): p. 2217-22). Finally pedicalled island flaps are wrapped aroundthe engineered tracheal transplant to provide a vascular source(Teramachi, M., et al., Intrathoracic tracheal reconstruction with acollagen-conjugated prosthesis: evaluation of the efficacy of omentalwrapping. J Thorac Cardiovasc Surg, 1997. 113(4): p. 701-11).Considering the extreme and urgent conditions under which theseengineered tracheas were transplanted, the strategy has shown modestsuccess, arguably more so with the decellularized allografts than withthe synthetic scaffolds based upon mortality rates to date. Further,despite the publication of clinical transplantation reports of thecurrent generation of engineered tracheas, the molecular and cellularprocesses controlling the survival of the engineered tracheal graftsremain incompletely defined. There is a need for superior decellularizedECM material, for example, decellularized trachea materials.

SUMMARY

Provided herein are methods of making decellularized ECM material. Thematerial is decellularized by application of a pressure differential tothe tissue to be decellularized, such as a cyclical pressuredifferential, resulting in an ECM material that is superior in manyinstances to ECM materials decellularized by other methods, such as byuse of boiling water, agitation, or acids, which can damage theend-product. According to certain embodiments, the tissue is placed in ahypertonic or a hypotonic solution that optionally contains one or moreof a detergent; an enzyme; and/or an acid, such as peracetic acid. Alsoprovided is a system or apparatus comprising a vacuum chamber includinga tissue cassette, optionally comprising a tissue sample to bedecellularized.

A method of decellularizing tissue is provided. The method compriseschanging a pressure at least one time to decellularize the tissuesample. The pressure is changed by any useful method, such as bychanging pressure within an airtight chamber. Thus, according to oneembodiment, the tissue sample is placed into an airtight chamber andpressure is changed in the container. An airtight container may be anysize, ranging, for example and without limitation, from a smallbench-top vacuum chamber, such as the chamber shown in connection withthe examples below, to a room-sized container, e.g., a room,facilitating bulk decellularization of large quantities of tissue. Inone embodiment, the tissue sample is immersed in a decellularizationsolution and is optionally agitated during application of the pressurechange. Non-limiting examples of a decellularization solution is anaqueous solution such as water, phosphate-buffered saline (PBS) orsaline. According to further embodiments, the solution further comprisesone or more of a surfactant, a salt, a sugar, an acid, a protease, or aDNAse, for example and without limitation the decellularization solutionis an aqueous solution comprising one or more of: SDS; CHAPS;deoxycholate; Triton X-100; Trypsin; DNAse; Proteinase K; NaCl; glucose;urea; or peracetic acid. In one embodiment, the decellularizationsolution comprises Triton X-100 and NaCl. According to one embodiment,the method further comprises after changing the pressure in the chamberat least one time, agitating the tissue in a solution comprisingperacetic acid and ethanol. In one embodiment, the pressure is changedcyclically with a frequency of from 5 seconds to one hour, for examplethe pressure is changed cyclically with a frequency of from 5 seconds to30 minutes and in one embodiment from 1 to 2 minutes. The duty cycle, aratio of the time it takes to pressurize or evacuate to how long apressure is held, ranges in one embodiment from 15% to 90%. In oneembodiment, relative pressure ramp rates, that is the rate of pressurechange during pressurization or evacuation, ranges from 0.25 MPa/s to0.0001 MPa/s. In one embodiment, the pressure is changed at least about10% or at least about 25%. For example a change of pressure from 1 atmto 2 atm is a 200% change, a change from 2 atm to 0.5 atm is a 75%change, a change of from 2 atm to 1 atm is a 50% change, and a change offrom 0.5 atm to 2 atm is a 400% change. The pressures may range above orbelow ambient, environmental pressure, such as above or below 1 atm or0.1013 MPa. In another embodiment, the pressure is changed at leastabout 0.10 MPa, or at least about 0.25 MPa. A typical absolute pressurerange is from 0.93 MPa to 0.006 MPa, and values and incrementstherebetween.

Also provided is a method of preparing an ECM material, comprisingdecellularizing tissue according to any method described herein, andsterilizing, packaging and/or drying, cryopreserving, freezing orlyophilizing the decellularized tissue. In one embodiment, the methodcomprises decellularizing the tissue and sterilizing, packaging anddrying, cryopreserving, freezing or lyophilizing the decellularizedtissue.

Also provided is a system for decellularization of tissue, comprising:an airtight chamber; a pump connected to the airtight chamber; one ormore valves for controlling air flow into and from the airtight chamber;and a computer control comprising one or more processes for changingpressure at least one time in the airtight chamber. In one embodiment,the airtight chamber comprises a tissue sample in a decellularizationsolution. In another embodiment, processes cyclically control pressurechange in the airtight chamber with a cycle length of from 15 seconds toone hour, a pressure change of at least 25%, and at least 10 pressurechanges.

In further embodiments, methods of preparing tissue, such as tracheatissue, are provided. The tissue is prepared by implanting thedecellularized tissue prepared according to any method described herein,in a patient. This can be used for trachea repair, by orthotopicallyimplanting decellularized trachea as described herein in a patient'sairway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Orthotopic transplantation or fresh tracheal grafts isassociated with superior recovery post operatively. Orthotopictransplants of fresh and decellularized tracheal grafts were performedon wild-type C57BL/6 mice. (FIG. 1A) Still frames from video recordingof tracheal transplant performed in a wild-type mouse. Survival (FIG.1B, fresh: n=7, decellularized: n=17) and weight gain (FIG. 1C, n>3 ateach time point) over four weeks following surgery shown.

FIG. 2-1 through 2-3. Histologic quantification of basal cell proteinexpression in mouse trachea transplants. Mice receiving fresh (a, b, e,f, i, j) or decellularized (c, d, g, h, k, l) transplants weresacrificed one (a-d), four (e-i), or eight (i-l) weeks following surgeryand immunofluorescent labeling for basal cell markers was performed.Images are shown at a site of anastomosis (a, e, i, c, g, k) or at amid-graft region (b, f, j, d, h l). Cells expressing keratin-5 (K5, red,m), keratin-14 (K14, green, n), and dual-expressing (yellow, o) cellsalong the basement membrane were counted in order to determine celldensities (cells/μm). Columns labeled “Ctrl” denote cell densities foruntreated, native trachea from C57BL/6 mice. Scale bar 200 μm.

FIGS. 3-1 through 3-3. Histologic quantification of ciliated andsecretory cell maturation in mouse trachea transplants. Mice receivingfresh (a, b, e, f, i, j) or decellularized (c, d, g, h, k, l)transplants were sacrificed one (a-d), four (e-i), or eight (i-l) weeksfollowing surgery and immunofluorescent labeling for markers of matureepithelial cells was performed. Images are shown at a site ofanastomosis (a, e, i, c, g, k) or at a mid-graft location (b, f, j, d, hl). Cells expressing acetylated tubulin (ACT, red, m) and Clara cellsecretory protein (CCSP, green, n) along the basement membrane werecounted in order to determine cell densities (cells/μm). Columns labeled“Ctrl” denote cell densities for untreated, native trachea from C57BL/6mice. Scale bar 200 μm.

FIGS. 4A and 4B. Ciliated cell function of orthotopic fresh anddecellularized tracheal transplants. Real time video microscopy wasemployed to quantify ciliary activity eight weeks after transplantation.Image from decellularized graft shown in FIG. 4A; calculated ciliarybeat frequencies shown in FIG. 4B. Column labeled “Ctrl” denotes ciliarybeat frequency for untreated, native trachea from C57BL/6 mice.

FIG. 5. Micro-computed tomography analysis of fresh orthotopic trachealtransplants. Representative images shown from computed tomographyperformed on explanted trachea from wild type, untreated C57BL/6 mice(a), mice receiving fresh transplants (b), and mice receivingdecellularized transplant (c).

FIG. 6. Decellularized tracheas maintain patency after eight weeks.

FIG. 7 is a diagram of components of one embodiment of a vacuum systemas described herein.

FIGS. 8A and 8B are graphs showing Failure Stress (FIG. 8A) and MaximumStiffness (FIG. 8B) as described in Example 3.

FIG. 9 is a graph showing Dynamic Viscosity versus Frequency, accordingto Example 4.

FIG. 10 is a schematic depiction of a computer.

FIG. 11 Top: Decellularized dermis stained with hematoxylin and eosin,bottom native porcine dermis.

FIG. 12 Comparing mechanical properties of native, vacuumdecellularized, and agitation decellularization control obtained from ascaled ASTM ball burst test. Top: Maximum stiffness calculated over a20% moving window. Bottom. Stress at failure.

FIG. 13 A: Hematoxylin and eosin stain of native porcine aorta. B.Hematoxylin and eosin stain of decellularized porcine aorta. C. DNA gelelectrophoresis of vacuum decellularized porcine aorta. First lane is a100-1200 base pair DNA ladder. Right hand lanes demonstrate remnant DNAfragment lengths of vacuum decellularized aorta are reduced to less than300 base pairs.

FIG. 14. 12 week explants of decellularized porcine aortas implanted ina 15 kg piglet partial circumference aortic reconstruction model.

DESCRIPTION OF THE INVENTION

Provided herein is a superior method for decellularizing tissue for usein regenerative medicine.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of ranges is intendedas a continuous range including every value between the minimum andmaximum values. As used herein “a” and “an” refer to one or more.

As used herein, the terms “extracellular matrix” and “ECM” refer to anatural scaffolding for cell growth. ECM, found in multicellularorganisms, such as mammals and humans, are complex mixtures ofstructural and non-structural biomolecules, including, but not limitedto, collagens, elastins, laminins, glycosaminoglycans, proteoglycans,antimicrobials, chemoattractants, cytokines, and growth factors. Inmammals, ECM often comprises about 90% collagen, in its various forms.The composition and structure of ECMs vary depending on the source ofthe tissue. For example, brain, trachea, vascular, cardiac, smallintestine submucosa (SIS), urinary bladder matrix (UBM), liver stromaECM, and dermal ECM each differ in their overall structure andcomposition due to the unique cellular niche needed for each tissue.

By “biocompatible”, it is meant that a device, scaffold composition,etc. is essentially, practically (for its intended use) and/orsubstantially non-toxic, non-injurous or non-inhibiting ornon-inhibitory to cells, tissues, organs, and/or organ systems thatwould come into contact with the device, scaffold, composition, etc.

In general, the method of preparing an ECM material requires theisolation of ECM from an animal of interest and from a tissue or organof interest. In certain embodiments, the ECM is isolated from mammaliantissue. As used herein, the term “mammalian tissue” refers to tissuederived from a mammal, wherein tissue comprises any cellular componentof an animal. For example and without limitation, tissue can be derivedfrom aggregates of cells, an organ, portions of an organ, orcombinations of organs. In certain embodiments, the ECM is isolated froma vertebrate animal, for example and without limitation, human, monkey,pig, cattle, and sheep. In certain embodiments, the ECM is isolated fromany adult, neonatal or fetal tissue of an animal, for example andwithout limitation, airway, trachea, vocal fold, lung, urinary bladder,liver, intestine, esophagus, pancreas, dermis, myocardium, heart valve,thoracic aorta, abdominal aorta, ocular such as retina, CNS such asbrain or spinal cord, peripheral vasculature, peripheral nerves,skeletal muscle and orthopedic sources such as nucleus polopsus,cartilage (e.g., knee and temporomandibular joint), tendon and bone.

As an example, and where applicable, the ECM is derived from tissue thatincludes all portions/components of the tissue from which it is derived,or less than all portions/components of that tissue source. For example,where applicable, the ECM may or may not include the basement membraneportion of the ECM. In certain embodiments, the ECM includes at least aportion of the basement membrane. The ECM may or may not retain some ofthe cellular elements that comprised the original tissue such ascapillary endothelial cells or fibrocytes.

As used herein, the term “derive” and any other word forms of cognatesthereof, such as, without limitation, “derived” and “derives”, refers toa component or components obtained from any stated source by any usefulmethod. For example and without limitation, an ECM-derived materialrefers to a material comprised of components of ECM obtained from anytissue by any number of methods known in the art for isolating ECM, forexample as described herein. In another example, mammaliantissue-derived ECM refers to ECM comprised of components of a particularmammalian tissue obtained from a mammal by any useful method.

Tissue for preparation of ECM materials can be harvested in a largevariety of ways and once harvested, a variety of portions of theharvested tissue may be used. For example and without limitation, in oneembodiment, the ECM is isolated from harvested porcine urinary bladderto prepare urinary bladder matrix (UBM). Excess connective tissue andresidual urine are removed from the urinary bladder. The tunica serosa,tunica muscularis externa, tunica submucosa and most of the muscularismucosa is removed in one embodiment, by mechanical abrasion or by acombination of enzymatic treatment, hydration, and abrasion. Mechanicalremoval of these tissues can be accomplished by abrasion using alongitudinal wiping motion to remove the outer layers (particularly theabluminal smooth muscle layers) and even the luminal portions of thetunica mucosa (epithelial layers). Mechanical removal of these tissuesis accomplished by removal of mesenteric tissues with, for example,Adson-Brown forceps and Metzenbaum scissors and wiping away the tunicamuscularis and tunica submucosa using a longitudinal wiping motion witha scalpel handle or other rigid object wrapped in moistened gauze.

In one embodiment, the ECM is prepared by abrading porcine bladdertissue to remove the outer layers including both the tunica serosa andthe tunica muscularis using a longitudinal wiping motion with a scalpelhandle and moistened gauze. Following eversion of the tissue segment,the luminal portion of the tunica mucosa is delaminated from theunderlying tissue using the same wiping motion. Care is taken to preventperforation of the submucosa. After these tissues are removed, theresulting ECM consists mainly of the tunica submucosa.

In another embodiment, dermal tissue is used as the source of ECM.Dermal tissue may be obtained from any mammalian source, such as human,monkey, pig, cow and sheep. In one embodiment, the source is porcine.Porcine skin from the dorsolateral flank of market weight pigsimmediately can be harvested and processed as described herein, and canbe delaminated to remove subcutaneous fat, connective tissue and theepidermis. The harvested sheets of porcine dermis can be immediatelyfrozen at −80° C. for storage.

In another embodiment, the epithelial cells are delaminated first byfirst soaking the tissue in a de-epithelializing solution such ashypertonic saline, for example and without limitation, 1.0 N saline, forperiods of time ranging from 10 minutes to 4 hours. Exposure tohypertonic saline solution effectively removes the epithelial cells fromthe underlying basement membrane. The tissue remaining after the initialdelamination procedure includes epithelial basement membrane and thetissue layers abluminal to the epithelial basement membrane. This tissueis next subjected to further treatment to remove the majority ofabluminal tissues but not the epithelial basement membrane. The outerserosal, adventitial, smooth muscle tissues, tunica submucosa and mostof the muscularis mucosa are removed from the remainingde-epithelialized tissue by mechanical abrasion or by a combination ofenzymatic treatment, hydration, and abrasion.

Methods described herein utilize pressure changes to enhance thedecellularization process for tissue. The pressure change can be appliedby any of a variety of methods. According to one non-limiting embodimentdepicted in FIG. 7, a vacuum chamber is connected to a solenoid, acryotrap and a vacuum pump via a solenoid or any other suitable valveconfiguration. The vacuum pump is used to change pressure in the vacuumchamber and the solenoid is electronically controlled by a PC (personalcomputer), or any other applicable computing device, such as a tablet orsmartphone. A vacuum or pressure can be applied by any of a multitude ofpossible configurations either understood or readily envisionable bythose of ordinary skill.

The pressure difference may be applied once or more than once atirregular or regular time periods. The timing for applying one or morepressure changes is infinitely variable, though variations ofexceptionally long periods may prove impracticable for commercial orother reasons. According to one example, the pressure change is appliedcyclically or regularly with a cycle period ranging from seconds, tominutes, to hours. In one embodiment, the number of cycles is at leastone. A cycle, or cycling, means a change of pressure in one direction(increase or decrease) from an original pressure followed by a change ofpressure in the opposite direction, for example and without limitation,returning to the original pressure. As an example, a vacuum (evacuation)is applied, followed by a pressurization; for example first decreasingpressure from an original value and subsequently increasing the pressureto the original value or substantially to the original value. In anotherexample, pressure is applied followed by evacuation; for example, firstincreasing pressure from an original value and subsequently returningthe pressure to the original value by evacuation. In another embodiment,the change in pressure in a cycle is from a first value to a secondvalue, higher or lower than the first value, and a return from thesecond value to the first value, or a change in pressure from the secondvalue towards (in the direction of) the first value. In one embodiment,the pressure in a cycle is raised or lowered from a first value to asecond value and then returned to the first value. For example, thecycle period may be 5, 10, 15, 20, 25, 30, 45 or 60 seconds, 2, 3, 4, 5,10, 15, 20, 30, 45 or 60 minutes, or 2, 3, 4, 5, 6, 9 or 12 hours or anyincrement therebetween. In one embodiment, the cycle time ranges from 5seconds to 30 minutes, in another, it ranges from 1 to 2 minutes, withthe vacuum chamber being evacuated between 50% and 90% of the time. Inone embodiment, relative pressure ramp rates, that is the rate ofpressure change during pressurization or evacuation, ranges from 0.25MPa/s to 0.0001 MPa/s. The duty cycle of the pressure/vacuum system is apercentage of time the system is held at a fixed pressure. Anon-limiting example of a duty cycle is 15% to 90% of cycle time at afixed pressure. The pressure may also be changed irregularly with thesame exemplary time limits. Although the pressure may be changed once,more typically the pressure is changed two or more and at least 5, 10,15, 20, 25, or 50 times, including increments therebetween. The pressurechange may be small, such as a difference of 1-5%, larger, such as adifference of 10-25%, or even larger ranging above 25% or 50%. Anexample differential may be between atmospheric pressure (1 atmosphereor ˜0.101 MPa (megapascal)) and any fraction or multiple thereof, suchas 0.001 or lower, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.095, 0.1, 0.125, 0.15, 0.25, 0.5 or 1 MPa. The pressure change may bebetween values above, and/or values below 1 atmosphere, so long as thereis a pressure change. For example, in one embodiment, both the maximumand minimum pressure are less than one atmosphere. In another, both themaximum and minimum pressure are above one atmosphere (positivepressure). In yet another embodiment, the minimum is below oneatmosphere and the maximum is above one atmosphere. As a non-limitingexample, the particular system depicted in FIG. 7 and described in theexamples below has a useful range of from −0.095 MPa to 0.415 MPa. Amaximum pressure change and/or maximizing the vacuum is preferred inembodiments. As would be understood by those of ordinary skill, thevalues, such as, without limitation, the cycle time, pressure ramprates, starting and ending pressures in a cycle, duty cycle, pressurehold times, and pressure change can be varied over the course of thedecellularization process.

During decellurization and during the application of a pressure change,the tissue to be decellularized is placed in a hypotonic, a hypertonicor an isotonic decellularization solution and is optionally agitate,e.g., on a shaker. The solution may be water, such as distilled,filtered or deionized water, PBS (phosphate-buffered saline), or saline.Surfactants, ionic or non-ionic, such as, without limitation, SDS(sodium dodecyl sulfate), CHAPS (e.g.,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), deoxycholateor Triton X-100 (e.g., 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethyleneglycol) optionally are included in the solution. Enzymes, such as,without limitation, Trypsin, DNAse or Proteinase K also optionally areincluded in the solution. Salts and sugars, such as, without limitation,NaCl, glucose, or urea optionally are included in the solution. An acid,such as peracetic acid (CH₃CO₃H) also may be included in the solution.

Decellularized ECM can then be dried, either lyophilized (freeze-dried)or air dried. The material is optionally sterilized, e.g., by gammaradiation or ethylene oxide exposure. The ECM is optionally comminuted.Dried ECM can be comminuted by methods including, but not limited to,tearing, milling, cutting, grinding, and shearing. The comminuted ECMcan also be further processed into a powdered form by methods, forexample and without limitation, such as grinding or milling in a frozenor freeze-dried state. As used herein, the term “comminute” and anyother word forms or cognates thereof, such as, without limitation,“comminution” and “comminuting”, refers to the process of reducinglarger particles into smaller particles, including, without limitation,by grinding, blending, shredding, slicing, milling, cutting, shredding.ECM can be comminuted while in any form, including, but not limited to,hydrated forms, frozen, air-dried, lyophilized, powdered, sheet-form. Inyet another embodiment, the ECM material, e.g., comminuted, is digestedwith a protease, and can be used for serum supplementation or in cellculture.

In one embodiment, the ECM is prepared from trachea tissue, for exampleand without limitation as described below. In use, the decellularizedECM may be affixed in place in the trachea, e.g., anamastosed by anyuseful manner, including without limitation, suturing or stapling.Alternately, anamastosis of ends of a trachea may be reinforced byoverlaying the trachea-derived ECM material over the anamastosis site.

In one embodiment, a method of preparing an ECM material in provided,comprising decellularizing tissue with a pressure-change step, accordingto any method described herein, and sterilizing, packaging (e.g., insuitable vessels, containers, foil and/or polymeric pouches, etc.according to acceptable packaging practices for implantable medicaldevices) and/or drying, cryopreserving, freezing or lyophilizing thedecellularized tissue. In one embodiment, the method comprisesdecellularizing the tissue, sterilizing, and drying, cryopreserving,freezing or lyophilizing the decellularized tissue.

As described above, a system is provided for decellularization oftissue, comprising: an airtight chamber; a pump connected to theairtight chamber; one or more valves for controlling air flow into andfrom the airtight chamber; and a computer control comprising one or moreprocesses for changing pressure at least one time in the airtightchamber. The airtight chamber is any suitable chamber, vessel,container, room, etc. capable of withstanding the varying pressures usedto prepare the tissue, which can be negative and/or positive pressuresin relation to atmospheric pressure. In one embodiment, the airtightchamber comprises a tissue sample in a decellularization solution. Inanother embodiment, processes cyclically control pressure change in theairtight chamber with a cycle length of from 15 seconds to one hour, apressure change of at least 25%, and at least 10 pressure changes. Anyvariation on pressure change parameters as described herein may beimplemented by the processes.

One of ordinary skill can devise and implement a suitable process forimplementing the computational framework by which pressure changes, suchas cyclical pressure changes in an airtight chamber may be implementedusing, e.g., a common pressure/vacuum pump and one or more valves, suchas one or more solenoids.

Processes are implemented on an electronic computing device (computer),e.g., by a microprocessor. In the context of computing, a process is,broadly speaking any computer-implemented activity that generates anoutcome, such as implementation of a mathematical or logical formula oroperation, algorithm, etc. FIG. 10 illustrates one embodiment of asystem 100 for implementing a modeling system. The system 100 mayinclude a device 102 operating under the command of a controller 104.Device 102 may be referred to herein, without limitation, as a computeror computing device. The broken lines are intended to indicate that insome implementations, the controller 104, or portions thereof consideredcollectively, may instruct one or more elements of the device 102 tooperate as described. Accordingly, the functions associated with theprocesses (e.g., software, programs) described herein may be implementedas software executing in the system 100 and controlling one or moreelements thereof. An example of a device 102 in accordance with oneembodiment of the present invention is a general-purpose computer orprocessor capable of responding to and executing instructions in adefined manner. Other examples include a special-purpose computerincluding, for example, a personal computer (PC), a workstation, aserver, a laptop computer, a web-enabled telephone, a web-enabledpersonal digital assistant (PDA), a microprocessor, an integratedcircuit, an application-specific integrated circuit, a microprocessor, amicrocontroller, a network server, a Java™ virtual machine, a logicarray, a programmable logic array, a micro-computer, a mini-computer, ora large frame computer, or any other component, machine, tool,equipment, or some combination thereof capable of responding to andexecuting instructions.

In one non-limiting embodiment, system 100 is implemented as a PC.Furthermore, the system 100 may include a central processing engineincluding a baseline processor, memory, and communications capabilities.The system 100 also may include a communications system bus to enablemultiple processors to communicate with each other. In addition, thesystem 100 may include storage 106 in the form of computer readablemedium/media, such as a disk drive, disk, optical drive, a solid statedrive, a tape drive, flash memory (e.g., a non-volatile computer storagechip), cartridge drive, and control elements for loading new software.In embodiments of the invention, one or more reference values may bestored in a memory associated with the device 102. Data, such as imagesproduced by the methods and systems described herein may be organized oncomputer readable media in a database, which is an organized collectionof data for one or more purposes, usually in digital form. In oneembodiment, any or all software, data, code, processes, controllers,algorithms, instructions, etc. are stored non-transiently on acomputer-readable medium.

Embodiments of the controller 104 may include, for example, a program,code, a set of instructions, or some combination thereof, executable bythe device 102 for independently or collectively instructing the device102 to interact and operate as programmed, referred to herein as“programming instructions”. One example of a controller 104 is asoftware application (for example, operating system, browserapplication, client application, server application, proxy application,on-line service provider application, and/or private networkapplication) installed on the device 102 for directing execution ofinstructions. In one embodiment, the controller 104 may be a Windows™based operating system. The controller 104 may be implemented byutilizing any suitable computer language (e.g., C\C++, UNIX SHELLSCRIPT, PERL, JAVA™, JAVASCRIPT, HTML/DHTML/XML, FLASH, WINDOWS NT,UNIX/LINUX, APACHE, RDBMS including ORACLE, INFORMIX, and MySQL) and/orobject-oriented techniques.

In one embodiment, the controller 104 may be embodied permanently ortemporarily in any type of machine, component, physical or virtualequipment, storage medium, or propagated signal capable of deliveringinstructions to the device 102. In particular, the controller 104 (e.g.,software application, and/or computer program) may be stored on anysuitable computer readable media (e.g., disk, device, or propagatedsignal), readable by the device 102, such that if the device 102 readsthe storage medium, the functions described herein are performed. Forexample, in one embodiment, the controller 104 may be embodied invarious computer-readable media for performing the functions associatedwith processes embodying the methods described herein.

In one embodiment, the software is written and developed using aprogramming language, such as the C++ computing language. An integrateddevelopment environment (IDE), such as “DevC++, may be used to write thecode. A person of ordinary skill in the art of computer programming andengineering will be able to develop and implement software capable ofcarrying out the tasks/operations described herein.

In use, the decellularized tissue described herein is orthotopicallyimplanted into a patient, such as a human patient. As an example, in thecase of a patient with a damaged or otherwise insufficient trachea,decellularized trachea tissue of an appropriate diameter that has beendecellularized by a method described herein in which pressure ischanged, is anastomosed to existing trachea in the patient.

EXAMPLE 1 Decellularized Tracheal Extracellular Matrix SupportsEpithelial Migration, Differentiation and Function

We tested the hypothesis that decellularized tracheal scaffolds promotecellular invasion/repopulation and functional epithelialization intransplanted tracheal grafts. Employing unique orthotopic murine and rattracheal transplant models we report the first evidence thatdecellularized tracheal scaffolds promote rapid functional cellularrestoration and provide a rationale for continued development of thistechnology.

Materials and Methods

Animals. A total of 60 female C57BL/6 mice (approximately 12 weeks, and20 g) were used in the study, half as donors and half as recipients.

Orthotopic tracheal transplantation. Recipient and donor animals wereanesthetized with intraperitoneal injections of ketamine (80 mg/kg)xylazine (8 mg/kg). Animals were positioned in a supine position andmaintained on a heating pad throughout the surgery. The trachealreconstruction was performed with microscopic assistance as describedpreviously (Hua, X., et al., Heterotopic and orthotopic trachealtransplantation in mice used as models to study the development ofobliterative airway disease. J Vis Exp, 2010(35); Genden, E. M., et al.,Orthotopic tracheal transplantation in the murine model.Transplantation, 2002. 73(9): p. 1420-5; Genden, E. M., et al., Thekinetics and pattern of tracheal allograft re-epithelialization. Am JRespir Cell Mol Biol, 2003. 28(6): p. 673-81; and Genden, E. M., et al.,Orthotopic tracheal allografts undergo reepithelialization withrecipient-derived epithelium. Arch Otolaryngol Head Neck Surg, 2003.129(1): p. 118-23). The ventral cervical trachea was exposed through amidline incision. The graft was then prepared by removing any looseconnective tissue from the surface and liquid from the lumen, and wascut to a length of five cartilaginous rings. Care was taken to maintainthe proximal-distal orientation of the grafts, particularly for thefresh transplants. A segment of three recipient tracheal rings wasdissected from the surrounding connective starting approximately fourrings below the larynx, with care not to damage the recurrent laryngealnerves. Once the tracheal segment was freed, a transverse cut was madein the intracartilaginous tissue until a complete transection wasperformed. A second transection was performed to remove two completerings of the trachea. Hemostasis was performed through the process. Thedistal anastomosis was performed first followed by the proximalanastomosis. In both cases, the anastomosis was performed with twointerrupted 10-0 Prolene sutures placed near the dorsal ends of thecartilage rings and one or two sutures placed on the ventral aspect ofthe tracheal repair. The strap muscles were re-approximated, and theskin incision was the closed with interrupted 7-0 PDS sutures. Animalswere allowed to recover from anesthesia. The operative time averaged 20minutes. Among the donor tracheas, half were harvested and immediatelyimplanted into a recipient. The other half were decellularized asdescribed below. Among the recipients, a segment of the native trachealwas excised. The recipients were separated into two groups, and thetrachea was reconstructed with either a fresh transplant or adecellularized trachea. Donor tracheas were placed in sterilephysiologic saline on ice until transported to the lab fordecellularization or until used for surgery (within approximately 20minutes when used for surgery).

Post-operative care. After surgery, mice were housed in groups of fourto five in standard cages, and food and water was supplied ad librum.The following medications were administered as subcutaneous injectionsfor five days following surgery: buprenorphine (0.1 mg/kg) twice dailyfor pain relief, gentamicin (8 mg/kg) once daily for infectionprophylaxis. At one, four, and eight weeks following surgery animalswere sacrificed with intraperitoneal injections of ketamine/xylazinefollowed by immediate exsanguination, and the tracheas were harvestedfor analysis.

Decellularization. Tracheas were trimmed of extra tissue under adissecting microscope (Zeiss StemiDV4) and were then frozen at −80° C.until time for surgery. The tracheas were thawed in deionized water atroom temperature. Tracheas were then decellularized with fourteen90-minute cycles each consisting of deionized water, then 3% TritonX-100, and then 3M NaCl treatments, leaving a decellularized trachealscaffold. During this process, tracheas were subjected to cyclicalpressure changes between room atmosphere and vacuum in a customapparatus. For this experiment, the chamber was evacuated from ˜0.1 MPato 0.02 MPa in 20 seconds, held for 20 seconds, and then was pressurizedfrom 0.02 MPa to ˜0.1 MPa in 20 seconds. Finally, the decellularizedscaffolds were agitated at 200 RPM on a shaker in a 0.1% peracetic acid(PAA)/4% ethanol solution for 90 minutes at 4° C. followed by three 30minutes rinses in phosphate buffered saline (PBS), shaken at 200 RPM at4° C. Scaffolds were then individually packaged in physiologic saline,and terminally sterilized by exposure to 20 kGy gamma irradiation.

Histology. Explants selected for histology were embedded in wax anddeparaffinized in two xylene washes, followed by rehydration in anethanol series. Antigen retrieval was performed using 10 mM citratebuffer in double distilled water. 5% bovine serum albumin in PBS wasused as a blocking reagent. For the keratin-5 (K5)/keratin-14 (K14) dualstains, the following primary antibodies were applied: mouse anti-K14(1:500 in blocking reagent) (Thermo/Neomarkers MS-115-P0) and rabbitanti-K5 (1:1000) (Covance PRB-160P). These primary antibodies weredetected with appropriate secondary antibodies: AlexaFluor488-conjugated goat anti-mouse IgG₃ (1:500) (Invitrogen A21151),AlexaFluor 594-conjugated donkey anti-rabbit (1:500) (Invitrogen A21207)Likewise, for acetylated tubulin (ACT)/Clara cell secretory protein(CCSP) dual stains, the following antibodies were applied: mouseanti-ACT IgG_(2b) (diluted 1:20000) (Sigma T6793) and goat anti-CCSP(1:1000) (kindly provided by Dr. Peter Di, University of Pittsburgh).These were detected with: donkey anti-mouse IgG (H+L) 594 (1:500)(Jackson Immuno 715-485-150), and donkey anti-goat IgG (H+L) 488 (1:500)(Jackson Immuno 715-515-150). All slides were counterstained withVectaShield Mounting Medium with DAPI (Vector Laboratories H-1200).Completed slides were examined with an Olympus IX71 florescencemicroscope (Nikon) and the images were captured with Nikon cellSensDimension (version 1.5). Adobe Photoshop CS5 was used to form a mosaicof all the images from a given slide for quantification.

Quantification. ImageJ (NIH, Bethesda, Md.) was used to measure thelength of several basement membrane segments along each explantedtracheal lumen. For each measured segment, DAPI-stained andantibody-immunolabeled cells along the segment were hand-counted inorder to determine cell densities (cells/μm). Mean cell densities werecalculated for each explanted trachea at each timepoint.

Cilia beat frequency evaluation. Three tracheas from each treatmentgroup were harvested eight weeks after surgery. Strips of trachealtissue were secured luminal side down on a 35-mm glass-bottomed culturedish (Willco Wells) using a glass coverslip covered with a siliconesheet containing a small window to form a chamber. Cilia dynamics werecaptured along the edge of the trachea strips at room temperature with a×100 differential interference contrast (DIC) oil objective and a Leicainverted microscope (Leica DMIRE2), and movies [200 frames/s (fps)] weremade with a Phantom v4.2 camera (Vision Research). To quantify ciliarybeat frequency (CBF), ImageJ was used to examine cyclic variations inpixel intensities corresponding to ciliary strokes. More than threerandomly selected areas were imaged from each of trachea in order tocalculate mean native and graft CBF for each treatment group.

Micro computed tomography. Three-dimensional image acquisition ofexplanted tracheas was carried out using a high resolution micro-CT(Siemens, Inveon Multimodality, Munich, Germany) at 12 μm imageresolution at 80 kVe and 500 μm X-ray. 3D surface volume rendering imagewas reconstructed using OsiriX software.

Statistics. Statistical analysis was performed using GraphPad Prism 6(GraphPad, La Jolla, Calif.). Data are presented as mean±one standarddeviation (SD) for each group. For weight change and survival analysis,linear regression and log-rank (Mantel-Cox) tests were performed,respectively. Differences in cell counts and ciliary beat frequenciesbetween untreated controls, fresh transplants, and decellularizedtransplants were assessed with two-way analysis of variance (ANOVA) withTukey's multiple comparisons test. Statistical significance was definedas p-value <0.05.

Results

Orthotopic tracheal transplantation rescues mice following trachealloss. Tracheal loss is often fatal, whereas lack of pre-clinical modelshas hindered tracheal replacement development (Grillo, H. C., Trachealreplacement: a critical review. Ann Thorac Surg, 2002. 73(6): p.1995-2004). We tested the hypothesis that orthotopic decellularizedtracheal transplant would rescue mice following full thickness trachealloss. We performed orthotopic transplantation using fresh anddecellularized grafts in age matched female mice as described.Representative images show excellent healing of the fresh trachealtransplant rescuing mice from full thickness tracheal loss (FIG. 1B,1C). In early studies, the survival rate for mice receiving freshtransplants was 71.4% (n=7) (FIG. 1B). Mean weight gain for survivinganimals was 15.1±3.4% over the 28-day period following surgery (FIG.1C).

Orthotopic transplantation of decellularized tracheal scaffolds rescuesmice from full thickness tracheal loss. Though freshly harvestedtracheal grafts successfully salvaged mice after acute tracheal loss, itwas not clear if similar results would be obtained using decellularizedtracheal scaffold. We decellularized adult murine tracheas achievingcomplete cellular removal. We rescued 35.3% (n=17) of mice from fulltracheal loss with decellularized tracheal transplants (FIG. 1B).Surviving animals lost weight over the 28-day study period, dropping to89.2±16.0% of their starting weight.

Decellularized tracheal scaffolds display epithelial restorationfollowing orthotopic transplantation. Our finding that decellularizedtracheal scaffolds rescued mice from full thickness tracheal defectssuggested that the decellularized scaffolds reconstituted the cellularpopulation. Immunofluorescent labeling of fresh and decellularizedgrafts post-transplant demonstrated complete resurfacing of the internalsurface of transplanted decellularized grafts (FIGS. 2-1 through 2-3,and 3-1 through 3-3). Time course quantification of cell specificrepopulation of the tracheal scaffold was performed. Though resurfacingof the decellularized tracheal scaffold lumen was complete within eightweeks (FIGS. 2-1 through 2-3, and 3-1 through 3-3), there were cell typespecific variations. Within the first week following surgery, weobserved rapid repopulation of the luminal surface with a large numberof keratin-5/keratin-14 dual-expressing (K5+/K14+) cells and a smallnumber of keratin-5 negative/keratin-14 positive (K5-/K14+) cells (FIGS.2-1 and 2-2, d, h, i). Over successive time-points the population ofK5+/K14+ cells steadily declined while the proportion of K5+/K14− andACT+ cells increased (FIG. 2-3, m-o, and FIG. 3-3, m). This same patternwas observed at the interface between native and graft tissue in freshtransplants (FIGS. 2-1 and 2-2, a, e, i). At eight weeks post-transplantthe regenerated epithelium contained similar numbers of K5+/K14+,K5−/K14+, and K5+/K14+ cells compared to fresh orthotopic trachealtransplants (FIG. 2-3, m-o). Total numbers of secretory cells permicrometer though initially not significantly different from numbers infresh transplants were decreased in decellularized grafts after eightweeks (FIG. 3-3, n). Cartilaginous portions of the decellularizedtrachea did not repopulate over the course of the study period.

Cilia function is diminished in decellularized tracheal transplants. Acritical feature of the healthy trachea is the ability to handlesecretory load (REF), and a robust ciliated cell response is a minimalrequirement for engineered tracheal transplants. We usedstate-of-the-art real-time microscopic imaging to assess cilia functionin our decellularized and fresh tracheal transplants (a movie wascaptured showing restoration of cilia function within decellularizedtracheas. Videos were captured at 200 frames/second, and exported at 30frames/second) and calculate ciliary beat frequency (CBF). We observedthe presence of functional cilia in both decellularized and freshorthotopic tracheal grafts. In both fresh and decellularized transplantgroups, CBF for cells along the graft was not significantly differentthan adjacent native tissue. However, ciliary beat frequency in bothfresh and decellularized grafts was significantly lower than that ofnative, untreated trachea (FIG. 4B).

Tracheal diameter is maintained in orthotopic tracheal transplants. Thecurrent generation of engineered tracheal transplants has beencomplicated by loss of structural integrity and an inability to maintaintracheal diameter (Kocyildirim, E., et al., Long-segment trachealstenosis: slide tracheoplasty and a multidisciplinary approach improveoutcomes and reduce costs. J Thorac Cardiovasc Surg, 2004. 128(6): p.876-82). We assessed tracheal morphology using micro-computed tomography(FIG. 5). Cartilaginous rings were visible as radiopaque structures infresh transplants (FIG. 5b ), as they were in wild-type untreatedcontrols (FIG. 5a ), but could not be visualized in decellularizedtransplants (FIG. 5c ).

Discussion

This example provides quantification and functional assessment of thefirst ever murine model of orthotopic, decellularized trachealtransplant. Long term survival was achieved in a significant majority ofcases (>70%) of fresh tracheal transplants and in a minority of cases indecellularized tracheal transplants; this difference trended towardsignificance at p=0.169. Transplant failures occurred from anastomosisseparation between the normal tracheal remnant and the transplant, andemphasize the need for care in placing and securing the back and frontwall sutures.

The trachea is potentially an ideal candidate for repair using adecellularized graft. A mature epithelium is a desirable component ofany tracheal graft, in order to (1) act as a barrier defense and (2) toprovide mucociliary clearance. It has been reported that a confluentepithelial layer can reduce or even prevent fibrosis and ultimatestenosis of a tracheal graft (Okumura, N., et al., Experimental study ona new tracheal prosthesis made from collagen-conjugated mesh. J ThoracCardiovasc Surg, 1994. 108(2): p. 337-45 and Teramachi, M., et al.,Intrathoracic tracheal reconstruction with a collagen-conjugatedprosthesis: evaluation of the efficacy of omental wrapping. J ThoracCardiovasc Surg, 1997. 113(4): p. 701-11). We observed completeresurfacing of the decellularized tracheal lumen by the end of the firstweek, following early proliferation of K5+/K14+ cells. The tracheallining is complex, and composed of heterogeneous cell populations, someof which act as progenitor cells (Musah, S., et al., Repair of trachealepithelium by basal cells after chlorine-induced injury. Respir Res,2012. 13: p. 107 and Cole, B. B., et al., Tracheal Basal cells: afacultative progenitor cell pool. Am J Pathol, 2010. 177(1): p. 362-76).Previous studies have demonstrated that K5+/K14+ cells represent aprecursor cell population with the capacity to develop into ciliated(ACT+) and secretory (CCSP+) cells (Cole, B. B., et al., 2010. 177(1):p. 362-76). This finding is supported by our histological data, whichshows a correlation between the depletion of the K5+/K14+ cellpopulation and the generation of a mature differentiated epithelium(FIGS. 2-1 through 2-3, and 3-1 through 3-3). Similarly, in animalsreceiving fresh transplants, the presence of K5+/K14+ cells near sitesof anastomosis suggests that this cell population plays a role inepithelial healing (FIG. 2-1, a). Real-time microscopy demonstrates thepresence of a functional ciliated epithelium eight weeks followingsurgery in both fresh and decellularized transplant groups (FIG. 4A).

Histologically, we observed that cartilaginous portions of thedecellularized trachea remain acellular throughout the course of thehealing period, while chondrocytes within the fresh tracheal transplantsare maintained. Computed tomography (FIG. 5) demonstrated radiolucentcartilaginous rings within decellularized grafts after eight weeks.Scans performed on decellularized grafts before transplantationsimilarly demonstrated a lack of visible radiopaque cartilage (data notshown). These findings suggest that cartilaginous rings lose theirmolecular structure during the process of decellularization, and are notrepopulated after transplantation. Despite the presumptive loss ofmechanical structure associated with the degradation of thecartilaginous rings, decellularized tracheal grafts maintained theirpatency over eight weeks (FIG. 6).

These data demonstrate this model to be a reliable pre-clinical platformfor research and capture the high-throughput aspect of mice whileharnessing the power of mutant murine models to test fundamentalquestions wound healing from a molecular perspective.

EXAMPLE 2 Additional Tissues

In addition to trachea, airway (trachea and vocal fold); esophagus;liver; small or large intestine; dermis; cardiovascular (myocardium,heart valve, and both thoracic and abdominal aorta); and ocular (retina)tissue have been similarly successfully processed. CNS (optic nerve,brain, spinal cord, peripheral nerve, dura mater); peripheralvasculature; orthopaedic (nucleus polposus, cartilage (TMJ, kneemeniscus), tendon, bone); skeletal muscle; pancreas; and lung tissue areprocessed in a similar manner.

EXAMPLE 3 Processing of Porcine Dermis and Porcine Aorta

Porcine dermis and aorta were prepared according to the followingprotocol. The tissue was prepared in 14 steps over 14 days, each stepcomprising treatment of the tissue in four separate solutions (DI water,3% triton X-100, 3M NaCl, and 2,000 KU DNAse I in 1M NaCl), eachtreatment comprising 30 one-minute cycles. The for each cycle, thechamber was evacuated from ˜0.1 MPa to 0.006 MPa in 30 seconds, held at0.006 MPa for 25 seconds, and then pressurized from 0.006 MPa to ˜0.1MPa in 5 seconds. Samples were rinsed at 200 RPM in a flask between daysfor 8 hours in PBS

FIG. 11: Top: Decellularized dermis stained with hematoxylin and eosin,bottom native porcine dermis.

FIG. 12. Comparing mechanical properties of native, vacuumdecellularized, and agitation decellularization control obtained from ascaled ASTM ball burst test. Top: Maximum stiffness calculated over a20% moving window. Bottom. Stress at failure. FIG. 12 demonstratesincreased retention of native mechanical properties duringdecellularization.

FIG. 13: A: Hematoxylin and eosin stain of native porcine aorta. B.Hematoxylin and eosin stain of decellularized porcine aorta. C. DNA gelelectrophoresis of vacuum decellularized porcine aorta. First lane is a100-1200 base pair DNA ladder. Right hand lanes demonstrate remnant DNAfragment lengths of vacuum decellularized aorta are reduced to less than300 base pairs.

FIG. 14. 12 week explants of decellularized porcine aortas implanted ina 15 kg piglet partial circumference aortic reconstruction model. Graftsare quickly incorporated into surrounding native tissue. Explants do notdemonstrate dilation, stenosis, or necrosis.

FIGS. 8A and 8B are graphs showing the superior mechanical properties,including Failure Stress (FIG. 8A) and Maximum Stiffness (FIG. 8B)indicating that ECM material prepared by vacuum decellularizationaccording to the methods described herein achieves superior mechanicalproperties as compared to prior agitation-only processes, with the addedbenefit that strong agitation, which disrupts fragile tissues, is notneeded.

EXAMPLE 4 Porcine Vocal Fold

Porcine vocal fold tissue was prepared essentially as described above.FIG. 9 is a graph showing dynamic viscosity.

EXAMPLE 5 Porcine Brain

Porcine vocal fold tissue was prepared essentially as described above,with 9 cycles each comprising 3 solutions(DI water, 3% Triton X-100, 3MNaCl) each comprising 30 one minute cycles: Cycle Pressure Ramp rate(evacuate chamber from ˜0.1 MPa to 0.014 MPa in 30 seconds, depressurize0.014 MPa to ˜0.1 MPa in 30 seconds).

The present invention has been described in accordance with severalexamples, which are intended to be illustrative in all aspects ratherthan restrictive. Thus, the present invention is capable of manyvariations in detailed implementation, which may be derived from thedescription contained herein by a person of ordinary skill in the art.

1-23. (canceled)
 24. A system for decellularization of tissue,comprising: a) an airtight chamber capable of withstanding varyingpressures; b) a decellularization solution enclosed within the airtightchamber; c) a vacuum pump connected to the airtight chamber capable ofcyclically varying pressure in the airtight chamber below or above oneatmosphere; d) one or more valves for controlling air flow into and fromthe airtight chamber; and e) a computer control comprising one or moreprocesses for changing pressure over at least one cycle in the airtightchamber.
 25. (canceled)
 26. The system of claim 24, in which thepressure change in the airtight chamber comprises a cycle length from 15seconds to one hour, a pressure change of at least 25%, and at least 10pressure changes.
 27. The system of claim 24 wherein the vacuum pump iscapable of cyclically changing pressure over at least one cycle in theairtight chamber with a pressure change ranging from 0.001 MPa to 1 MPa.28. The system of claim 24 wherein the decellularization solutioncomprises a substance selected from the group consisting of a detergent,a surfactant, a sugar, a salt, an acid, a protease, and a DNAse.
 29. Thesystem of claim 24 wherein the decellularization solution comprises asubstance selected from the group consisting of SDS, CHAPS,deoxycholate, Triton X-100, trypsin, proteinase K, glucose, urea,peracetic acid, and ethanol.
 30. The system of claim 24 wherein thepressure is changed cyclically with a frequency of 5 seconds to 30minutes.
 31. The system of claim 24 further comprising an agitator. 32.The system of claim 24 wherein the tissue is selected from the groupconsisting of brain, vascular, cardiac tissue, small intestine, urinarybladder, liver, skin, vocal cord, esophagus, large intestine, aorta,ocular, optic nerve, spinal cord, peripheral nerve, dura mater,cartilage, tendon, bone, nucleus polposus, skeletal muscle, pancreas,lung, amnion, chorion, placenta, and trachea tissue.
 33. The system ofclaim 24 wherein the rate of pressure change in the airtight chamberduring pressurization or evacuation ranges from 0.25 MPa/s to 0.0001MPa/s.
 34. The system of claim 24 further comprising a lyophilizer. 35.The system of claim 24 wherein said computer control comprises acomputing device and a controller executable by the device forperforming instructions associated with varying pressure in the airtightchamber by the vacuum pump and controlling airflow into the airtightchamber by the one or more valves.